Optogenetic tool for rapid and reversible clustering of proteins

ABSTRACT

A protein construct including a gene encoding a light-sensitive protein fused to at least one of either a low complexity sequence, an intrinsically disordered protein region (IDR), or a repeating sequence of a linker and another gene encoding a light-sensitive protein. Among the many different possibilities contemplated, the protein construct may also advantageously include cleavage tags. This protein construct may be utilized for a variety of functions, including a method for protein purification, which requires introducing the protein construct into a living cell, and inducing the formation of clusters by irradiating the construct with light. The method may also advantageously include cleaving a target protein from an IDR, and separating the clusters via centrifuge. A kit for practicing in vivo aggregation or liquid-liquid phase separation is also included, the kit including the protein construct and a light source capable of producing a wavelength that the light-sensitive protein will respond to.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.62/347,677, filed Jun. 9, 2016; 62/362,889, filed Jul. 15, 2016; and62/424,924, filed Nov. 21, 2016, which are herein incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. DA040601awarded by the National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The Sequence Listing filed electronically herewith is also herebyincorporated by reference in its entirety (File Name:PRIN-50505-SL_ST25.txt; Date Created: Jul. 8, 2019; File Size: 14,931bytes).

BACKGROUND OF THE INVENTION

Cellular function relies on coordinating the thousands of reactions thatsimultaneously take place within the cell. Cells accomplish this task inlarge part by spatio-temporally controlling these reactions usingdiverse intracellular organelles. In addition to classic membrane-boundorganelles such as secretory vesicles, mitochondria and the endoplasmicreticulum, cells harbor a variety of membrane-less organelles. Most ofthese are abundant in both RNA and protein, and are referred to asribonucleoprotein (RNP) bodies. Among dozens of examples include nuclearbodies such as nucleoli, Cajal bodies, and PML bodies, and cytoplasmicgerm granules, stress granules and processing bodies ((Mao et al.,2011), (Anderson and Kedersha, 2009), (Buchan and Parker, 2009),(Handwerger and Gall, 2006)). By impacting a number of RNA processingreactions within cells, these structures appear to play a central rolein controlling the overall flow of genetic information, and are alsoincreasingly implicated as crucibles for protein aggregation pathologies((Li et al., 2013), (Ramaswami et al., 2013)).

From a biophysical standpoint, these structures are remarkable in thatthey have no enclosing membrane and yet their overall size and shape maybe stable over long periods (hours or longer), even while theirconstituent molecules exhibit dynamic exchange over timescales of tensof seconds (Phair and Misteli, 2000). Moreover, many of these structureshave recently been shown to exhibit additional behaviors typical ofcondensed liquid phases. For example, P granules, nucleoli, and a numberof other membrane-less bodies will fuse into a single larger sphere whenbrought into contact with one another ((Brangwynne et al., 2009),(Brangwynne et al., 2011), (Feric and Brangwynne, 2013)), in addition towetting surfaces and dripping in response to shear stresses. Theseobservations have led to the hypothesis that membrane-less organellesrepresent condensed liquid states of RNA and protein that assemblethrough intracellular phase separation, analogous to the phasetransitions of purified proteins long observed in vitro by structuralbiologists ((Ishimoto and Tanaka, 1977), (Vekilov, 2010)). Consistentwith this view, RNP bodies and other membrane-less organelles appear toform in a concentration-dependent manner, as expected for liquid-liquidphase separation ((Brangwynne et al., 2009), (Weber and Brangwynne,2015), (Nott et al., 2015), (Wippich et al., 2013), (Molliex et al.,2015)). These studies suggest that cells can regulate membrane-lessorganelle formation by altering proximity to a phase boundary. Movementthrough such an intracellular phase diagram could be accomplished bytuning concentration or intermolecular affinity, using mechanisms suchas posttranslational modification (PTM) and nucleocytoplasmic shuttling.

Recent work has begun to elucidate the molecular driving forces andbiophysical nature of intracellular phases. Weak multivalentinteractions between molecules containing tandem repeat protein domainsappear to play a central role ((Li et al., 2012), (Banjade and Rosen,2014)). A related driving force is the promiscuous interactions (e.g.electrostatic, dipole-dipole) between segments of conformationallyheterogeneous proteins, known as intrinsically disordered protein orintrinsically disordered regions (IDP/IDR, which are typically, althoughnot necessarily, also low complexity sequences, LCS). Hereinafter, theterms intrinsically disordered protein, intrinsically disordered region,and intrinsically disordered protein region are used interchangeably.RNA binding proteins often contain IDRs with the sequence compositionbiased toward amino acids including R, G, S, and Y, which comprisesequences that have been shown to be necessary and sufficient fordriving condensation into liquid-like protein droplets((Elbaum-Garfinkle et al., 2015), (Nott et al., 2015), (Lin et al.,2015)). The properties of such in vitro droplets have recently beenfound to be malleable and time-dependent ((Elbaum-Garfinkle et al.,2015), (Zhang et al., 2015), (Weber and Brangwynne, 2012), (Molliex etal., 2015), (Lin et al., 2015), (Xiang et al., 2015), (Patel et al.,2015)), underscoring the role of IDR/LCSs in both liquid-likephysiological assemblies and pathological protein aggregates.

Despite these advances, almost all recent studies rely primarily on invitro reconstitution, due to a lack of tools for probing protein phasebehavior within the living cellular context. However, a growing suite ofoptogenetic tools have been developed to control protein interactions inliving cells. The field has primarily focused on precise control overhomo- or hetero-dimerization ((Toettcher et al., 2011), (Kennedy et al.,2010), (Levskaya et al., 2009)). But recent work suggests the potentialof optogenetics for studying intracellular phases, demonstrating thatlight-induced protein clustering can be used to activate cell surfacereceptors (Bugaj et al., 2013), as well as to trap proteins intoinactive complexes ((Lee et al., 2014), (Taslimi et al., 2014)).

To date, no platform has been provided which can be used to dynamicallymodulate intracellular protein interactions, enabling the spatiotemporalcontrol of phase transitions within living cells. Such a platform wouldbe highly desirable.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to protein constructs with the abilityto induce and control reversible liquid-liquid phase separation, bothglobally and at specific subcellular locations. This system reveals thatthe location within the phase diagram can be used to dictate thematerial state of phase-separated IDR clusters, ranging from dynamicliquid droplets to arrested but reversible gels, which can over timemature into irreversible aggregates.

In the present invention, systems and methods are provided that utilizeprotein constructs with at least two regions fused to each other: (i) alight sensitive region containing a first segment (e.g., a proteinsensitive to at least one wavelength of light) and (ii) a functionalregion containing a second segment (e.g., a low complexity sequence(LCS), an intrinsically disordered protein region (IDR), or one or morerepeatable sequences).

Among the many different possibilities contemplated, a protein constructmay also advantageously contain a desired protein to purify, or afluorophore. In some embodiments, the second segment is an IDR, wherethe IDR is a portion of FUS, Ddx4, or hnRNPA1. In some embodiments, theprotein sensitive to at least one wavelength of light used in the firstsegment contains a protein that is sensitive to visible light. In someembodiments, the protein sensitive to at least one wavelength of lightused in the first segment is Cry2, Cry2olig, PhyB, PIF,light-oxygen-voltage sensing (LOV) domains, or Dronpa. It iscontemplated that these protein constructs will be configured such thatafter being introduced into a living cell, typically throughtransfection with DNA encoding for the protein construct, which is thentranslated into the protein by the native cellular machinery, exposingthe living cell with the protein construct to certain wavelengths oflight will induce the protein constructs within the living cell tocluster. It is further contemplated that if these protein constructscontain cleavage tags, such as self-cleaving tags, Human Rhinovirus 3CProtease (3C/PreScission), Enterokinase (EKT), Factor Xa (FXa), TobaccoEtch Virus Protease (TEV), or Thrombin (Thr), then after a firstinduction, it may be advantageous to cleave and induce clustering again.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B illustrate two embodiments of protein constructs.

FIG. 2A is a graph of assembly rates for various protein constructs,including constructs without a light-sensitive region (101),Cry2olig-only constructs (102), optoFUS constructs (103), andCry2olig-FUS_(N) constructs (104).

FIG. 2B is a graph of disassembly rates for various protein constructs,including Cry2olig-only constructs (112), Cry2olig-FUS_(N) constructs(113), and optoFUS constructs (114).

FIGS. 3A and 3B are graphs of the temporal evolution of backgroundconcentrations outside clusters, C_(bg), for certain cells, where theactivation interval is T=30 s (FIG. 3A) or T=180 s (FIG. 3B). Dotsdenote time points when cell images were taken and dashed linesrepresent time points for blue light activation.

FIG. 3C is a graph indicating the steady-state background fluorescenceintensities of individual cells (open circles) under the givenactivation interval increase linearly (solid lines) with totalconcentration of protein constructs, as well as best fit lines forinterval times of T=6 s (210) and T=30 s (220).

FIG. 3D is a graph indicating the impact of activation interval on thesteady-state fractions of inactivated protein constructs for 4 differentamounts of 488 nm blue light (0.07 μW, 0.3 μW, 0.92 μW, and 2.4 μW);solid lines denote a global fit to data using the kinetic model.

FIG. 3E is a depiction of the calculated steady-state concentrations ofactivated protein constructs, showing a clear concentration thresholdfor light-mediated clustering, and a dotted horizontal line indicatingthe saturation concentration.

FIG. 4 is a schematic diagram illustrating the broadening effects ofslower inactivation kinetics and lower C_(sat) on the localized phasetransition, and specifically the localized phase transitions given fastinactivation kinetics and high C_(sat) (260) and the localized phasetransition given slow inactivation kinetics and low C_(sat) (262).

FIGS. 5A and 5B are images of the distinct morphology of phase separatedoptoFUS clusters for shallow (5A) and deep (5B) supersaturation.

FIG. 6 is a graph of FRAP recovery curves of optoFUS clusters formedwith varying supersaturation depths, including shallow quenching depths(310), intermediate quenching depths (320), and deep quenching depths(330).

FIG. 7 illustrates an embodiment of protein construct using repeatableunits.

FIG. 8A is an illustration of a two-component system leading tolight-activatable repeats, where a first component (810) includes fourtandem copies of a GCN4 peptide (812) separated by linkers (814), whichare fused to a fluorophore (816), and a second component (820) withfunctional region (822) that includes scFV-GNC4, a fluorophore (824) anda light sensitive region (826) that includes Cry2.

FIG. 8B illustrates the self-assembled construct shown in FIG. 8A in theabsence of activating light.

FIG. 8C is an illustration showing the resulting clusters of proteinconstructs upon light activation.

FIG. 8D illustrates several NIH3T3 cells, some of which only expressmCh-10XGCN4 but not scFV-GNC4-sfGFP-Cry2 (red cells), while othersexpress both constructs (yellow cells).

FIG. 8E illustrates that upon light activation, yellow cells expressingboth constructs exhibit light-activated clustering, while red cellsexpressing only mCh-10XGCN4 do not show any clustering.

FIG. 9A is an illustration of an example 3D phase diagram as a functionof the concentration of a particular repeat number for illustrationpurposes showing PhyB_(N)+PIF_(M), and apparent binding affinity,controlled by the 650/750 nm light ratio.

FIG. 9B is another illustration of a phase diagram, showing the processthat (i) occurs with a shallow quenching depth (250), resulting in theformation of a liquid droplet (251); and (ii) occurs with a deepquenching depth (252), resulting in the formation of solid-like gels(253) which can lead to the formation of irreversible aggregates (254).

FIG. 10 is a graph of a normalized number of clusters for four differentconstructs_during the first, second, or third cycle of quenching,including (i) constructs with Crt2WT but without FUS_(N) (410) duringfirst cycle; (ii) FUS_(N)-Cry2olig constructs (420) during first cycle;(iii) OptoFUS constructs with shallow quench depths (430) during firstcycle; and (iv) OptoFUS constructs with deep quench depths (440) duringfirst cycle, and (v) OptoFUS constructs with deep quench depths (440)during second cycle (442).

FIG. 11 is a flowchart depicting a method for clustering proteinconstructs and protein purification.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in further detail, it is to beunderstood that the invention is not limited to the particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, a limitednumber of the exemplary methods and materials are described herein.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise.

FIG. 1A depicts a generalized embodiment of the present invention. Aprotein construct (10) will typically comprise a light sensitive region(20) and a functional region (30). The construct may also optionallycomprise a first cleavage tag (40), a target protein (50), or a secondcleavage tag (60). Typically, the second cleavage tag (60) will not beneeded unless the construct is followed by another domain. The specificarrangement shown in FIG. 1A just serves as an example and otherpermutated arrangements can be employed.

The light sensitive region (20) typically includes a first segmentcomprising at least one protein sensitive to at least one wavelength oflight. In preferred embodiments, this segment includes Cry2, Cry2olig,PhyB, PIF, light-oxygen-voltage sensing (LOV) domains, or Dronpa. Inother embodiments, the segment includes a protein sensitive to a visiblewavelength of light, typically including wavelengths from about 400 nmto about 800 nm.

The functional region (30), which is fused to the light sensitive region(20), may include a second segment, the second segment comprising one ormore low complexity sequences, one or more intrinsically disorderedprotein regions (IDRs), one or more synthetic or natural nucleic acidbinding domains, or at least one repeatable sequence, the repeatablesequence comprising a linker fused to at least one additional geneencoding at least one protein sensitive to at least one wavelength oflight. In preferred embodiments, the protein construct comprises an IDR,where the IDR is a portion of a first protein selected from the groupconsisting of FUS (SEQ ID NO.: 3), Ddx4 (SEQ ID NO.: 4), and hnRNPA1(SEQ ID NO.: 5). In some embodiments, the IDR comprises amino acids1-214 of FUS, 1-236 of Ddx4, or 186-320 of hnRNPA1.

FIG. 1B shows an alternate arrangement of the protein construct,comprising an optional fluorophore (70). In FIG. 1B, the fluorophore(70) is fused between the light sensitive region (20) and the functionalregion (30), although other locations may be advantageous. In preferredembodiments, the fluorophore is mCherry, although the use of otherfluorescent proteins is also envisioned, including but not limited toGFP variants.

An example of the protein construct was produced by fusing the “sticky”IDR from various proteins to the photolyase homology region (PHR) ofArabidopsis thaliana Cry2, a light-sensitive protein which is known toself-associate upon blue light exposure. This IDR-Cry2 fusion proteinwould recapitulate the modular domain architecture of many phaseseparating proteins, but confer tunable light-dependence to itsmultivalent interactions.

The approach of replacing the multi-valent interaction domains of FUSand other IDR-containing RNA binding domains with a light-activatableCry2(WT) motif is inspired by native mechanisms utilized by cells tocontrol intracellular phase transitions. Phase separation in cellsappears to be regulated in two distinct but complementary ways: 1)changing the concentration of protein constructs, for example by proteintranslation, altered degradation, or nucleocytoplasmic shuttling, and 2)changing their intermolecular interaction strengths, for example throughPTMs, particularly phosphorylation which deposits a negative charge onS, T, or Y residues, which are commonly found in IDRs driving phaseseparation. Indeed, FUS is found in stress granules, one type ofmembrane-less body whose assembly depends on PTMs and proteinconcentration, and which has been suggested to assemble by regulatedintracellular phase separation.

DNA fragments encoding IDRs of human FUS (residues 1-214) and humanhnRNPA1 (residues 186-320) were amplified by PCR using FUS cDNA(GeneCopoeia, GC-F0952) and pET9d-hnRNP-A1 (Addgene plasmid #23026),respectively. A gene for the IDR of human dx4 (residues 1-236) wassynthesized (Integrated DNA Technologies). Sequences for mCherry andCry2olig (Addgene plasmid #60032) were cloned into the pHR lentiviralbackbone to generate the pHR-mCh-Cry2olig plasmid. A site-directedmutagenesis was then performed to produce the Cry2WT version. ForIDR-fusion Cry2 plasmids, DNA fragments encoding the IDRs were insertedinto the linearized pHR-mCh-Cry2WT (or Cry2olig) backbone usingIn-Fusion Cloning Kit (Clontech). The resulting constructs were fullysequenced to confirm the absence of unwanted substitutions.

These constructs were introduced into living cells. mCherry-labeled Cry2PHR (hereafter: Cry2WT) was first expressed in NIH 3T3 cells along witha few other variants. NIH 3T3 cells were cultured in 10% FBS (AtlantaBiological) in DMEM (Gibco) supplemented with penicillin, streptomycinand GlutaMAX (Gibco) at 37° C. with 5% CO₂ in a humidified incubator. Toproduce stable cell lines expressing cry2 fusion constructs, lentiviralconstructs were transfected with FuGENE (Promega), following themanufacturer's recommended protocol, into 293T cells that had beenplated in the 6-well dishes 1 day prior to the transfection. Viralsupernatants were collected 2 d after transfection and passed through a0.45-μm filter to remove cell debris. NIH 3T3 cells plated at ˜70%confluency in the 6-well dishes were infected by adding 0.4-1 ml offiltered viral supernatant directly to the cell medium. Viral medium wasreplaced with normal growth medium 24 h after infection.

The cells were then induced to cluster with blue light. Consistent withprevious reports, Cry2WT alone showed little clustering upon blue lightactivation. Strikingly, fusing the N-terminal IDR of FUS (FUS_(N)) toCry2WT (hereafter optoFUS) leads to rapid blue-light dependent clusterassembly in most cells. Similar results were seen upon fusing theC-terminal IDR of the ALS-related RNA binding protein hnNRNPA1(optoHNRNPA1), or the N-terminal IDR of Ddx4 (optoDDX4), both of whichhave been reported to drive liquid-liquid phase separation.

As shown in FIG. 2A, the optoFUS construct (103) exhibits ˜9-fold fasterassembly of clusters than Cry2olig constructs (102), under similarexpression level and activation conditions. Referring to FIG. 2B, it isalso noted that the optoFUS constructs (114) disassemble ˜3-fold fasterthan Cry2olig constructs (112). Interestingly, the assembly of Cry2oligclusters is also dramatically enhanced when it is fused to FUS_(N) (FIG.2A, element 104). However, the disassembly of these FUS_(N)-Cry2oligclusters (113) is now much slower than for Cry2olig alone (112).Moreover, these clusters (113) do not completely dissolve, even after >1hour without light activation.

Liquid droplets tend to adopt round shapes due to surface tension.Consistent with this feature of liquids, optoFUS clusters have roundmorphologies. A second feature common to liquid phase droplets is thatthe protein constructs within undergo dynamic exchange with thesurrounding solution. Fluorescence recovery after photobleaching (FRAP)experiments, involving bleaching the mCherry signal, shows a nearlycomplete recovery of the fluorescence signal, with a recovery time scaleof 140±10 s. Finally, in non-biological systems, small phase separateddroplets can dissolve at the expense of larger droplets, an effect knownas Ostwald ripening. Ostwald ripening is frequently observed in theoptoFUS clusters, particularly within those that assemble within thecell nucleus. Together, these data strongly suggest that optoFUSclusters formed upon blue light exposure are liquid phase droplets.

These liquid-like behaviors suggest that optodroplet assembly mayrepresent light-inducible liquid-liquid phase separation within thecell. A light-induced increase in Cry2 self-association affinity couldrepresent a controllable change to the effective valency of theconstructs. In the presence of light, each FUS-Cry2 fusion protein canassociate with other monomers through Cry2-Cry2 or FUS-FUS interactions,whereas only FUS-FUS interactions would be possible in the dark. In thisphysical picture, the light-increased avidity would result in thecrossing of a phase boundary and consequent initiation of liquid-liquidphase separation.

The concentration of light-activated optoFUS can be changed using atleast two independent methods: 1) by changing the total concentration ofoptoFUS protein constructs within the cell, and 2) by changing bluelight intensity. If the assembly mechanism is liquid-liquid phaseseparation, then droplet formation should depend on both optoFUSconcentration and light activation level. Consistent with this, dropletformation shows a strong dependence on blue light activation intensity.For an activation protocol which begins at a very weak power, initiallyno cells exhibit droplets, even after continuous weak blue lightactivation for 16 min. However, when the blue light power was tripled,those cells which express high levels of the optoFUS construct nowassemble droplets. Distinct and spatially separated droplets slowlynucleate and then grow in size; qualitatively, this behavior is verysimilar to the well-known nucleation and growth regime observed inshallow-quench phase transitions—i.e., in systems which are onlymoderately supersaturated.

The dependence of droplet assembly kinetics on the total concentrationof optoFUS was also tested. Blue light power was fixed and assembly wasexamined in cells with different expression levels; intracellularoptoFUS concentrations ranging from about 0.2 to about 13 μM were used,comparable to the estimated intracellular concentration of endogenousFUS: ˜1-10 μM. Consistent with the results obtained for varyingblue-light activation, the lowest expressing cells do not form dropletsat all. Interestingly, for cells that do form droplets, the higher theexpression level, the faster the assembly kinetics. Thus, both the totaloptoFUS concentration and blue light intensity collectively affectlight-induced droplet formation.

Finally, it was determined if the opposing effects from these twoparameters can compensate each other to give rise to similar assemblydynamics. Indeed, higher expressing cells exposed to weaker blue lightshow similar clustering kinetics as lower expressing cells exposed tostronger light. Taken together, these data suggest that theconcentration of light activated optoFUS can be used for controllingdroplet formation.

A simple kinetic framework for measuring the concentration of activatedprotein constructs, and its relationship to the onset of dropletcondensation, was developed to quantitatively test whether this systemreflects light-controllable phase separation. We assume that theinactivated state undergoes a first-order reaction to the activatedstate, with a reaction rate proportional to light intensity, accordingto k₁=k_(act)*[blue], where k_(act) is an activation rate constant and[blue] is the intensity of activating blue light. The activated proteinconstructs can also convert back to the inactivated state, at a rategiven by k₂. In this model, blue light exposure increases theconcentration of activated, self-associating protein constructs, whichdrives global phase separation upon exceeding the saturationconcentration, i.e., when C_(act)>C_(sat).

A series of activation protocols were employed with different activationintervals and blue light power. Cycles of light followed by dark wereutilized, since this provides the ability to probe both the activationrate constant, k_(act), and the inactivation rate constant, k₂. WhenoptoFUS cells are exposed to a pulse train of activating light stimuliwith high enough intensity, cells form droplets typically after a shortlag phase. As protein constructs are recruited into droplets, thebackground concentration (fluorescence intensity) outside of dropletsdecreases. As shown in FIGS. 3A and 3B, when the interval betweenconsecutive pulses is relatively long (e.g., T=180 s, shown in FIG. 3B)cycles of partial assembly and disassembly are observed, but forintervals shorter than 1 min (e.g., T=30 s, shown in FIG. 3A) thebackground intensity exhibits a monotonic decay to a steady state,Cbg,st.

In the simplest phase transition model, the steady-state backgroundconcentration is equal to a sum of the concentration of inactivatedprotein constructs, Cinact,st, and the activated protein constructsoutside clusters, C_(sat). Expressing C_(inact,st) as a fractionF_(inact,st) of the total concentration:C_(inact,st)=F_(inact,st)C_(tot), the steady-state backgroundconcentration is thus C_(bg,st)=F_(inact,st)C_(tot)+C_(sat). Consistentwith this model prediction, the steady-state background concentration ofactivated optoFUS cells increases linearly with total concentration.Moreover, as shown in FIG. 3D, varying activation intervals yielddifferent slopes (F_(inact,st)), but converge to a similar y-intercept(C_(sat), corresponding to ˜1.4 μM), consistent with the saturationconcentration representing an intrinsic property of the optoFUSconstruct. Indeed, the identical activation protocol, when applied tooptoDDX4 cells, yields 2-fold lower C_(sat), implying strongerintermolecular interaction between Ddx4 IDRs.

This kinetic framework can be utilized to quantify the rate constantsfor activation. We first computed the steady-state fraction ofinactivated protein constructs for each cell using the relationship,F_(inact,st)=(C_(bg,st)−C_(sat))/C_(tot), and the measured saturationconcentration. FIG. 3C shows the fraction of inactivated proteinconstructs for intervals of T=6 s and T=30 s, as well as best fit linesfor T=6 s (210) and T=30 s (220). In agreement with the modelpredictions, this illustrates that the fraction of inactivated proteinconstructs increases with either longer activation intervals or weakerblue light intensity. This can be well-fit to the functional dependencepredicted by the model, yielding values for the rate constants,k_(act)=7.4±4.7 μW⁻¹s⁻¹ and k₂=0.011±0.005 s⁻¹. Moreover, this alsoagree with the model prediction that at high enough power, theinactivated fraction becomes independent of blue light intensity, sinceall protein constructs already populate the activated state. Finally,phase separation should only occur if the total concentration ofactivated protein constructs exceeds the saturation concentration, Csat.This prediction is in good agreement with data for this embodiment,which show a sharp concentration threshold for the activated proteinconstructs, below which no cytoplasmic clusters were observed (See FIG.3E).

The preceding experiments and theoretical analysis show that fusing, forexample, self-associating IDRs to the light activation domain of Cry2WTenables light-activated phase separation. However, it is also possibleto modulate the assembly dynamics by changing the light activationdomain. Previously, a point mutant version of Cry2 (E490G), known asCry2olig, was shown to exhibit significant clustering The assembly ofCry2olig is also dramatically enhanced when it is fused to FUSN,exhibiting ˜9-fold faster assembly under similar expression level andactivation conditions, comparable to the rapid assembly of the optoFUSconstruct (i.e. FUSN-Cry2WT).

Applying the same method of cycled light activation described above, itwas found that there is also a saturation concentration ofFUSN-Cry2olig. However, the saturation concentration of FUSN-Cry2olig is5-fold lower than optoFUS, consistent with the point mutation (E490G) inCry2olig increasing homo-interaction strength. Moreover, theinactivation rate of FUSN-Cry2olig is 5-fold slower than optoFUS,consistent with the previous findings. Thus, utilizing IDR fusions withvarious other self-associating optogenetic proteins can be used to tunethe dynamics of light-induced intracellular phase separation.

While the above example utilizes IDRs, the functional region may alsoutilize other proteins, such as synthetic or natural nucleic acidbinding domains. Many RNA binding proteins contain self-associating IDRsor LCSs that can drive phase separation. However, additional RNA bindingdomains can enhance phase separation via multivalent interactions withRNA. For example, FUS is an ALS-related RNA binding protein involved indiverse nucleic acid processing including DNA repair, transcription andpre-mRNA splicing. While the self-associating N-terminal IDR of FUS hasbeen shown to be necessary and sufficient for liquid-liquid phaseseparation, C-terminal RNA binding domains appear to further promotephase separation. In preferred embodiments, the synthetic or naturalnucleic acid binding domains utilizes RNA recognition motifs (RRM),double-stranded RNA binding domains (dsRBD), S1, zinc finger bindingdomains, YT521-B homologies (YTH), DNA and RNA helicase domains,Pumilio, or S-adenosylmethionine (SAM) structures.

Rapid growth and fast inactivation lead to localized phase separation.Local changes in molecular interaction strength can induce intracellularphase separation at specific subcellular locations, as in the case of Pgranule condensation during C. elegans embryo development. Bycontrolling the spatial distribution of blue light, analogous localphase separation is achievable. When the corners of individual optoFUScells were locally illuminated, droplets rapidly assembled near theactivation zone, with a wave of droplet assembly propagating outward,but only over a short range near the activation zone. This was verifiedwith single line activation, localized in time and space. When a linepulse was applied to optoFUS cells, droplets immediately form along theactivation line. The width of cluster distribution was maintained over anarrow band, before all droplets began disassembling within a fewminutes.

To quantitatively elucidate the dynamics of phase separation uponlocalized activation, a simplified coarse-grained model was developedthat is consistent with a mesoscale model. This model describes theconcentration of activated protein constructs, c(x), as well as thedroplets they nucleate, which are characterized by the single fieldvariable θ_(d)(x,t) that represents the volume fraction of dropletswithin a given spatial volume. The model predicts that the steady-statedroplet profile width for continuous localized activation is given by:x₀ ^(SS)˜√{square root over (D/k₂)}ln[k₁E/(c_(sat)√{square root over(D(k₁+k₂))})], indicating that the primary factor is thereaction-diffusion length scale, √{square root over (D/k₂)}, where D isthe molecular diffusion coefficient in cytoplasm. Thus, diffusion ofactivated monomers will tend to oppose localized droplet formation,while rapid reversion to the dark state would sharpen dropletlocalization patterns. Numerical simulations of the model support thisphysical picture by reproducing the evolution time and extent ofexperimentally-observed droplet profiles, provided heterogeneous(seeded) nucleation kinetics are employed; interestingly, the observedbehaviors are not consistent with homogeneous nucleation.

This coarse-grained model predicts that the 5-fold slower inactivationrate (k₂) and 5-fold lower c_(sat) exhibited by FUSN-Cry2olig relativeto optoFUS would limit the ability to localize droplet assembly (SeeFIG. 4, compare diameter of localized phase transition 260 vs localizedphase transition 262). Consistent with the model prediction, inFUSN-Cry2olig cells, clusters first rapidly appeared at the localizedactivation zone, but a wave of cluster formation then propagated slowlyacross the entire cell; a single line pulse activation also displayed abroader cluster distribution than for optoFUS. Moreover, cellsexpressing Cry2olig alone exhibited a long lag time, followed by theconcomitant appearance of clusters even far away from the activationzone. These data demonstrate that localized phase separation seen inoptoFUS depends on the rapid growth conferred by the IDR, combined withthe relatively fast inactivation kinetics of Cry2WT.

The location within the phase diagram provides a degree of control overmaterial properties and aging potential of clusters that are induced. Insimple non-biological systems, quenching deep into the two phase region,corresponding to a high degree of supersaturation, can lead tocondensation of assemblies with arrested dynamics, typically referred toas gels or glasses. Similar arrested dynamics can be observed in livingcells, by exposing cells with similar expression levels to varying bluelight intensity, thus moving into different depths beyond the phaseboundary. For shallow quenching, cells typically showed no clusteringduring a long lag period of ˜100 seconds, followed by slow phaseseparation. As the quenching depth increases, the lag period shortens;for sufficiently high blue light activation, phase separation isinitiated immediately after activation. Notably, as shown in FIGS. 5Aand 5B, while shallow quenching (FIG. 5A) tends to give rise to therelatively round droplet-like assemblies such as those described above,deep quenching (FIG. 5B) leads to the formation of structures withhighly irregular shapes. Small diffraction-limited puncta that appearedimmediately upon blue light exposure grew in size over time, in largepart due to sticking to one another, forming highly branched elongatedstructures. Consistent with the apparent gel-like nature of theseassemblies, FRAP measurements reveal that the major fraction of proteinconstructs within these clusters do not exchange with the surroundingcytoplasm. Indeed, as shown in FIG. 6, as the quenching depth increasesfrom shallow (310) to intermediate (320) to deep (330), the fraction ofrecovery decreases, implying an increase in the solid fraction. Thus,the material state of light-activated assemblies can be tuned bycontrolling the cytoplasmic location within the phase diagram.

The assembly of structures such as P granules, Ddx4 puncta, and nucleolialso appear to be controlled through a combination of PTMs and proteinconcentration levels, which would similarly allow cells to move theircytoplasm into different regions of a high-dimensional phase diagram.

Referring again to FIG. 1, when cleavage tags are utilized, at least onecleavage tag (40) is typically inserted between the functional regionand a protein that has been targeted for, e.g., purification. A widevariety of cleavage tags are envisioned, including but not limited to:self-cleaving tags, Human Rhinovirus 3C Protease (3C/PreScission),Enterokinase (EKT), Factor Xa (FXa), Tobacco Etch Virus Protease (TEV),and Thrombin (Thr).

The functional region may also utilize a repeatable element. As shown inFIG. 7, the functional region can comprise repeatable units of a linkerfused to a gene encoding a protein sensitive to light, where the numberof repeats is typically, although not limited to, 1 to 20. Preferablythe number of repeats is from 2 to 9, and more preferably the number ofrepeats is about 4.

Although many variants are envisioned, FIGS. 8A-C depict one exampleinvolving a first protein (810) construct which may have, for example, 4tandem copies (812) of the GCN4 peptide (SEQ ID NO.: 1:EELLSKNYHLENEVARLKK), each separated by linkers (814), which is thenfused to a fluorophore (816). A second protein construct (820) maycomprise scFV-GNC4-sfGFP-Cry2, where the functional region comprisesscFV-GNC4 (822), sfGFP (824) acts as a fluorophore, and the lightsensitive region comprises Cry2 (826). As depicted in FIG. 8B, withoutlight, the second protein construct binds to the peptide binding siteson the first protein construct. Upon exposure to light, Cry2-Cry2binding occurs, creating large clusters of the first and second proteinconstructs, shown in FIG. 8C.

FIG. 8D illustrates several NIH3T3 cells, some of which only expressmCh-10XGCN4 but not scFV-GNC4-sfGFP-Cry2 (red cells), while othersexpress both constructs (yellow cells). FIG. 8E illustrates that uponlight activation, yellow cells expressing both constructs exhibitlight-activated clustering, while red cells expressing only mCh-10XGCN4do not show any clustering, consistent with the fact that clustering forthe system in the example above would require Cry2 multimerization.

At least three protein construct system configurations are alsoenvisioned that utilize multiple, different protein constructscomprising repeatable units. In the first configuration, a system isenvisioned wherein the light sensitive regions are identical, but thenumber of repeating units is different. An example of this is aCry2(-linker-Cry2)_(n) arrangement, where the system contains threetypes of constructs, where n=2, 5, and 12. Although preferredembodiments utilize between about 2 and about 12 repeatable units, it isenvisioned that a protein construct can utilize any number of repeatableunits.

In the second configuration, at least two types of constructs are used,each having the same number of repeating units, but having differentlight sensitive regions. In a preferred embodiment, the system uses twotypes of constructs, each comprising at least a portion of one of a pairof proteins, such as Cry2-CIB, PhyB-PIF, or iLID-SspB.

This second configuration is based on the recognition that by changingthe affinity and valency of protein-protein interactions we can controlboth the phase behavior and properties of the resulting droplets. ThePhyB/PIF optogenetic system is able to change interaction affinities byvarying the ratio of 650 nm and 750 nm light applied to the PhyBprotein. The range of achievable interaction affinities is broadlytunable—individual PhyB-PIF interactions are very weak under pure 750 nmlight (>100 μM), but very strong under pure 650 nm light (<100 nM). Bychanging the 650/750 nm ratio, any intermediate affinity can beattained. By using PhyB and PIF constructs with different number ofrepeats, the multivalency can be further tuned to induce phaseseparation under even modest concentrations (<1 μM). Otherlight-activatable proteins may also be used, such as the PHR domain ofthe protein Cry2. When activated with blue light (488 nm), thesemultimerized Cry2 constructs will phase separate into droplets orgel-like structures. This enables building light induced clusters,either within living cells or in vitro, with potential applications fromperturbing intracellular organization, to purifying proteins throughfusion with affinity purification tags and centrifuging the phaseseparated droplets.

In the case where the light activatable domains are PhyB/PIF pairs, onecan express and purify in E. coli (BL21) polymers (e.g., 5-mers) of polyPhyB (PhyB₅), and 5-mers of poly PIF (PIFS). It is also advantageous toinclude, for example, a TEV-cleavable His-tag, although other knownmethods for cleaving are envisioned.

These constructs can then be mixed, and illuminated with defined ratiosof 650/750 nm light from computer-controlled LED sources. Lower repeatnumbers, e.g. PhyB₃ and PIF₃, as well as higher repeat numbers, e.g.PhyB₁₀ and PIF₁₀ may also be used. Moreover, it is possible to use mixedrepeat number solutions, i.e., PhyB_(M)+PIF_(N), where M≠N underdifferent light activation settings (650/750 nm), to optimize formaximum optical control of assembly under different physiologicalprotein concentrations ([PhyB_(M)], [PIF_(N)]≤μM); using where M≠Nallows for selective sequestration. The precise concentrations andrepeat number of the various constructs, together with the degree oflight activation, allows for control over the phase behavior andproperties of the resulting assembles, which may be liquid like or moresolid like, as shown in the example schematic in FIG. 9A.

Conversion of molecular species from weak self-association state to highself-association one, for example through post-translationalmodification or exposure of RNA in RNP complexes, leads to liquid-liquidphase separation. As shown in FIG. 9B, when the depth is shallow, thisprocess follows the green path (250) to produce liquid droplets (251).Deep supersaturation along the red path (252) results in the formationof solid-like gels (253), with arrested molecular dynamics. Gels areinitially reversible, but slow dynamics within promote the formation ofirreversible aggregates over time (254).

Disassembly dynamics of these constructs, upon turning off blue light,has also been tested. As shown in FIG. 10, Without FUS_(N), Cry2WT (410)only forms noticeable clusters in a small subset of cells, but thesedisassemble relatively quickly and show no irreversibility even aftermultiple activation cycles. By contrast, under the same conditions(shallow quench), the optoFUS construct (430) forms clusters whichdisassemble at a rate roughly 3 fold slower than clusters seen in Cry2WTalone (410); this indicates that the self-associating FUS_(N) chainsinteract with sufficient strength to delay dissolution. Gel-like optoFUSclusters assembled from deep quenching (440) also shrink in size uponturning off blue light, while maintaining their overall irregularmorphology, and appear to be completely dissolved by ˜20 min.Interestingly, despite their reversibility after the first activationcycle, these gel-like clusters disassemble ˜1.4 times slower thanliquid-like optoFUS droplets (shallow quench) (430), suggesting thematerial state of clusters impacts the disassembly rate.

When cells expressing optoFUS undergo a sequence of repeated cycles ofdeep quenching (440), some clusters appear to remain as early as the endof the second cycle (442). By the third cycle, roughly 20% of clusterswere not fully dissolved. Concomitantly, the disassembly rate ofgel-like clusters gradually slowed down over subsequent cycles. Whentested up to five cycles, the number of remaining clusters increasesprogressively for each cycle. These aggregates are truly irreversible:after the cessation of light activation cycles, they remain assembledfor at least 6 hours. Remarkably, no irreversible clustering is observedin liquid-like optoFUS clusters, formed through cycles of shallowquenching (430). One interpretation for these results is that deepquenches develop irreversible aggregates simply because more materialhas assembled into each cluster. However, even when the total amount ofphase separated material is smaller than cells with liquid droplets,cycles through the gel state robustly accumulate irreversibleaggregates, confirming that the gel state specifically promotesirreversible aggregate formation.

The gel state provides a crucible for promoting irreversible aggregateformation. This irreversibility is reminiscent of observations ofclusters remaining after just a single round of assembly in cellsexpressing FUS_(N)-Cry2olig. Since FUS_(N)-Cry2olig clusters form gelsregardless of quenching depth, prolonged incubation of proteinconstructs in the gel state due to the slow inactivation rate ofCry2olig may be enough to induce irreversible aggregate formation evenfrom a single round of quenching.

Dynamically tuning protein interactions with light achieves high degreeof control over intracellular phase space, which can be exploited tostudy the phase diagram of FUS-mediated assemblies within living cells.Varying the degree of quenching depth leads to clusters spanningdifferent material states, ranging from liquid droplets to gels. Shallowquenching leads to liquid droplets, similar to those observed with FUSand other proteins both in vitro and in vivo. However, deep quenchingresults in the formation of gels, which exhibit minimal moleculardynamics and highly irregular aggregate-like morphologies. Theseassemblies are reminiscent of gel-like structures previously observed invitro for a variety of globular proteins. Notably, lysozyme, awell-folded protein whose phase behavior has been extensively studied invitro, exhibits liquid-liquid phase separation at modestsupersaturation, but upon deep quenching exhibits phase separation whoseprogress is arrested, with the condensed material forming a solid-likegel network. The gel state appears to represent a kinetically trappedstate arising from the slow relaxation between strongly interactingprotein constructs, rather than a thermodynamically favored state. Overtime, such gels can develop into crystals and fibers.

This suggests that increasing the strength or effective valency ofmolecular self-association (e.g., through light activation orendogenously through PTMs) can lead to liquid-liquid phase separation,or for higher supersaturation can result in gelation. It is known thatmembrane-less organelles can exhibit at least partially solid-likeproperties (i.e., viscoelasticity). Indeed, large variations in theimmobile fraction of stress granule proteins are often measured in FRAPexperiments, and in some cases stress granules begin to resembleirregularly shaped gels. These apparent differences in material statereflect different depths into the cytoplasmic phase diagram. Thisability to tune material states by moving within the phase diagram couldbe exploited by cells, since highly dynamic liquid-like states may beuseful as microreactors, while gel-like structures would provide anideal storage environment. However, assembling such arrested, gel-likestructures deep within the phase diagram comes with the danger ofproducing potentially toxic species, due to irreversible aggregation andfibrillization.

A method for protein purification, utilizing these constructs, isillustrated in FIG. 11. As described above, the method (500) generallycomprises at least seven steps: providing the protein construct (510) asdescribed above, where the protein construct also comprises a targetprotein (see, e.g., FIG. 1, element 50). The construct is expressed(520) in cells. Cells are then lysed (532) and cellular debris isremoved. The induction step (534) is followed by centrifugation toremove molecules other than the clustered protein construct. A cleavingstep (536) follows, where the target protein is cleaved from, forexample, an intrinsically disordered protein. After cleaving, a secondinduction step (538) is utilized. The second induction is typically forseparating the desired protein to be purified from the remainingportions of the protein constructs. After the second induction step(538), separation (539) can occur, typically via centrifuge.

When a goal is, for example, to purify molecules interacting with thetarget proteins, the method may be modified slightly. The living cell isexposed to at least one wavelength of light that the proteins sensitiveto at least one wavelength of light are responsive to, causing theprotein constructs to cluster which inherently induces molecules withinthe living cell that interact with the target protein to cluster (530).The induction step can modify at least one of the transport orreactivity of enzymes and other molecules within the living cell, and/orcause intermolecular interactions, protein activation or inactivation,manipulation of signaling pathways, or gene expression through theinduction of membrane-less bodies. Cells are gently lysed (540) and theinduced clusters are then separated (550), typically via centrifuge, andthe separated molecules are then purified (560) using typical proteinpurification methodologies.

In some embodiments, an induction step may also lead to nucleatingdroplets of tunable viscoelasticity at defined genomic loci, using atleast one of LacO arrays or dCas9. The engineered dCas9 with peptiderepeats, for example GCN4 peptide (SEQ ID NO.: 1: EELLSKNYHLENEVARLKK)or GFP11 (SEQ ID NO.: 2: RDHMVLHEYVNAAGIT), is co-expressed with aconstruct comprising the first segment of peptide-binding protein,either scFV-GNC4 or GFP1-10, and the second segment of FUS IDR.Coexpressing sgRNAs programmed for targeting specific genomic locidelivers dCas9 complexes with FUS IDR to the loci, which serves as aseed for droplet assembly. The viscoelasticity of droplets is tunedusing the similar strategy described above, a varying degree ofsupersaturation.

The platform can also facilitate catalytic activity uponphoto-stimulation by locally concentrating enzymes inside or outsidecells, for instance for intracellular production of natural products,biofuels etc. This may be accomplished by, for example, recruitingenzymes into the phase separated environment generated by the clusteringof intrinsically disordered protein regions.

Kits may also be provided to simplify the use of these methods. The kitswill generally include a protein construct as described above, as wellas at least one light emitting device that can be used to activate thelight sensitive proteins of the protein construct.

Thus, specific constructs and methods which can be used for, e.g., rapidand reversible clustering of proteins, have been disclosed. It should beapparent, however, to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of thedisclosure. Moreover, in interpreting the disclosure, all terms shouldbe interpreted in the broadest possible manner consistent with thecontext. In particular, the terms “comprises” and “comprising” should beinterpreted as referring to elements, components, or steps in anon-exclusive manner, indicating that the referenced elements,components, or steps may be present, or utilized, or combined with otherelements, components, or steps that are not expressly referenced.

What is claimed is:
 1. A method for forming irreversible aggregateswithin a cell, comprising the steps of: a) providing a cell withpolynucleic acid encoding a protein construct, wherein the proteinconstruct comprises: a first segment comprising at least one proteinsensitive to at least one wavelength of light; and a second segmentfused to the first segment, the second segment comprising at least oneintrinsically disordered protein region (IDR), and wherein said firstsegment and said second segment are heterologous; b) culturing the cellunder conditions which will result in the expression of the proteinconstruct within the cell; and c) inducing the protein construct tocluster and form an irreversible aggregate by repeatedly exposing theprotein construct within the cell to the at least one wavelength oflight.
 2. The method according to claim 1, wherein inducing the proteinconstruct to cluster changes the physiology of the cell by modifyingtransport or reactivity of molecules with the cell.
 3. The methodaccording to claim 1, wherein inducing the protein construct to clusterchanges physiology of the cell by causing intermolecular interactions,protein activation or inactivation, manipulation of signaling pathways,or gene expression clusters within the cell.
 4. The method according toclaim 1, further comprising lysing the cell and separating theirreversible aggregate via centrifuge.
 5. The method according to claim1, wherein the protein construct further comprises a cleavage tag. 6.The method according to claim 5, wherein the cleavage tag is selectedfrom the group consisting of: Human Rhinovirus 3C Protease(3C/PreScission), Enterokinase (EKT), Factor Xa (FXa), Tobacco EtchVirus Protease (TEV), and Thrombin (Thr).
 7. The method according toclaim 5, wherein the cleavage tag is a sell-cleaving tag.
 8. The methodaccording to claim 1, further comprising expressing a LacO array ordCas9 in the cell.
 9. The method according to claim 1, wherein the IDRis FUS.
 10. The method according to claim 1, wherein the IDR is Ddx4.11. The method according to claim 1, wherein the TDR is hnRNPA4.
 12. Themethod according to claim 1, wherein the at least one protein sensitiveto at least one wavelength of light is Cry2.
 13. The method according toclaim 1, wherein the at least one protein sensitive to at least onewavelength of light is Cry2olig.
 14. The method according to claim 1,wherein the at least one protein sensitive to at least one wavelength oflight is PhyB.
 15. The method according to claim 1, wherein the at leastone protein sensitive to at least one wavelength of light is PIF. 16.The method according to claim 1, wherein the at least one proteinsensitive to at least one wavelength of light is a light oxygen voltagesensing (LOV) domain.
 17. The method according to claim 1, wherein theat least one protein sensitive to at least one wavelength of light isDronpa.